KR20210059405A - Fusion protein comprising modified interleukin-7 and interleukin-2 and use thereof - Google Patents

Abstract

The present invention relates to a fusion protein comprising modified interleukin (IL)-7 and interleukin-2 and uses thereof. The fusion protein comprising the modified IL-7 and IL-2 of the present invention has a high production yield, induces differentiation of cytotoxic T cells, and can promote proliferation. In addition, the fusion protein of the present invention can promote the differentiation of NK cells and effectively inhibit cancer. Therefore, the fusion protein of the present invention can be usefully used for the treatment of cancer or infectious diseases.

Description

Fusion protein containing modified interleukin-7 and interleukin-2, and uses thereof {FUSION PROTEIN COMPRISING MODIFIED INTERLEUKIN-7 AND INTERLEUKIN-2 AND USE THEREOF}

The present invention relates to a fusion protein comprising modified interleukin-7 and interleukin-2, and uses thereof.

Interleukin 2 (hereinafter, IL-2) is a glycoprotein that plays an important role in the generation, survival and homeostasis of lymphocytes. The IL-2 is composed of 133 amino acids and mediates its action by binding to the IL-2 receptor (IL-2 Receptor) consisting of three or less individual subunits. IL-2 is mainly synthesized by activated T cells, especially CD4+ helper T cells. IL-2 stimulates the proliferation and differentiation of T cells, and induces the generation of cytotoxic T lymphocytes (CTL) and the differentiation of peripheral blood lymphocytes into CTL and Lymphokine-activated killer (LAK) cells. In addition, IL-2 induces the proliferation and differentiation of B cells, and promotes the synthesis of immunoglobulins by B cells. Furthermore, IL-2 induces the generation, proliferation and activation of natural killer cells (NK cells).

However, IL-2 is not only involved in the increase and activation of immune cells, but also has a dual function in that it maintains immune tolerance, and it has been reported that IL-2 is not optimal for inhibiting tumor growth. have. In addition, various IL-2 mutations have been studied to improve the therapeutic efficacy of IL-2 (US 5,229,109 B), but there are still many problems to be resolved in order to utilize IL-2 pharmacologically. .

On the other hand, interleukin-7 (interleukin 7, hereinafter, IL-7) is a cytokine that promotes an immune response via B cells and T cells, and particularly plays an important role in the adaptive immune system. Specifically, IL-7 activates immune functions through the survival and differentiation of T cells and B cells, the survival of lymphoid cells, and the activation of natural killer cells (NK cells). It is important for the development of cells and B cells. It binds to HGF (hepatocyte growth factor) and is a cofactor of pre-pro-B cell growth-stimulating factor and V(D)J rearrangement of T-cell receptor beta (TCRβ). ) (Muegge K, 1993, Science 261 (5117): 93-5).

However, when the recombinant IL-7 is produced for medical use, there is a problem in that a large amount of impurities is produced compared to a general recombinant protein, it is easy to denature, and it is not easy to mass-produce.

US 5,229,109 B

Muegge K, 1993, Science 261 (5117): 93-5

An object of the present invention is to provide a fusion protein comprising a modified interleukin-7 (IL-7) and interleukin-2 (IL-2).

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer or infectious diseases comprising the fusion protein as an active ingredient.

In order to achieve the above object, an aspect of the present invention is a modified interleukin-7 (IL-7) and interleukin-2 (IL-2) in which an oligopeptide consisting of 1 to 10 amino acid residues is bound to IL-7. It provides a fusion protein comprising a.

In another aspect of the present invention, a fusion protein comprising a modified interleukin-7 (IL-7) and interleukin-2 (IL-2) in which an oligopeptide consisting of 1 to 10 amino acid residues is bound to IL-7 is effective. It provides a pharmaceutical composition for preventing or treating cancer or infectious disease comprising as an ingredient.

The fusion protein comprising the modified IL-7 and IL-2 of the present invention has a high production yield, can induce differentiation of cytotoxic T cells, and promote proliferation. In addition, the fusion protein of the present invention promotes the differentiation of NK cells and can effectively inhibit cancer. Therefore, the fusion protein of the present invention can be usefully used in the treatment of cancer or infectious diseases.

1 is a diagram showing the IL7-hyFc-IL2v gene construct.
2 is a view showing the results of analysis by SDS-PAGE after purification of the IL7-hyFc-IL2v fusion protein.
3 is a diagram showing the results of analysis by SDS-PAGE and SE-HPLC after formulation of the IL7-hyFc-IL2v fusion protein.
4 is a diagram showing the proliferation rate of CD8+ T cells treated with anti-CD3ε antibody, IL-7-hyFc or IL7-hyFc-IL2v fusion protein by passage number.
5 is a diagram showing the expression levels of GzmB, IFN-gamma, and TNF-alpha in CD8+ T cells treated with anti-CD3ε antibody, IL-7-hyFc or IL7-hyFc-IL2v fusion protein.
6 is a view confirming the differentiation ability of CD8 T cells in the spleen of mice administered with PBS, IL-7-hyFc or IL7-hyFc-IL2v fusion protein.
7 is a diagram illustrating the size of cancer and the proliferation rate of NK cells after administration of PBS, IL-7-hyFc or IL7-hyFc-IL2v fusion protein in a mouse transplanted with a colon cancer cell line.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention provides a fusion protein comprising a modified interleukin-7 (IL-7) and interleukin-2 (IL-2).

The modified IL-7 may have the following structure:

A-IL-7;

In this case, A is an oligopeptide consisting of 1 to 10 amino acid residues, and the modified IL-7 is IL-7 or a polypeptide having similar activity.

The term "IL-7 or a polypeptide having similar activity" as used herein refers to a polypeptide or protein having the same or similar sequence and activity as IL-7.

The IL-7 may include an IL-7 protein or a fragment thereof. At this time, IL-7 may be derived from human, white mouse, mouse, monkey, cow, or sheep.

Specifically, human IL-7 may have an amino acid sequence represented by SEQ ID NO: 1 (Genbank Accession No. P13232); Rat IL-7 is a Genbank Accession No. May have the amino acid sequence disclosed in P56478; Mouse IL-7 is a Genbank Accession No. It is disclosed in P10168 and may have an amino acid sequence; Monkey IL-7 is a Genbank Accession No. It may have an amino acid sequence disclosed in NP_001279008; Bovine IL-7 is Genbank Accession No. May have the amino acid sequence disclosed in P26895; Yang IL-7 is Genbank Accession No. It may have an amino acid sequence disclosed in Q28540.

The IL-7 may be a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 1. In addition, the modified IL-7 is the sequence of SEQ ID NO: 1 and about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% or more may be the same.

In addition, the IL-7 protein or fragment thereof may contain variously modified proteins or peptides, ie, variants. The modification may be performed through a method of substituting, deleting or adding one or more proteins to wild-type IL-7, which does not alter the function of IL-7. These various proteins or peptides are wild-type proteins and 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99 It can have% homology.

In general, substitution of wild-type amino acid residues may be performed by alanine, or by conservative amino acid substitutions that do not affect or weaken the charge of the entire protein, that is, polarity or hydrophobicity.

Refer to Table 1 below for conservative amino acid substitutions.

Basic Arginine (Arg, R)
Lysine (Lys, K)
Histidine (His, H) acid Glutamic acid (Glu, E) Aspartic acid (Asp, D) Uncharged polar Glutamine (Gln, O) Asparagine (Asn, N)
Ser, S
Threonine (Thr, T)
Tyrosine (Tyr, Y) Non-polar Phenylalanine (Phe, F) Tryptophan (Trp, W)
Cysteine (Cys, C)
Glycine (Gly, G)
Alanine (Ala, A)
Valine (Val, V)
Proline (Pro, P)
Methionine (Met, M)
Leucine (Leu, L)
Norleucine
Isoleucine

For each amino acid, additional conservative substitutions include "homologs" of the amino acids. At this time, the "homolog" refers to an amino acid in which a methylene group (CH ₂ ) is inserted into the side chain of the beta position of the side chain of the amino acid. Examples of such "homologs" include, but are not limited to, homophenylalanine, homoarginine, homoserine, and the like.

The term "IL-7 protein" used in the present invention is also used as a concept including "IL-7 protein and fragments thereof". The terms “protein”, “polypeptide” and “peptide” may be used as interchangeable concepts unless otherwise specified.

In the structure of the modified IL-7, A may be directly linked to the N-terminus of the IL-7 or linked through a linker. The A may be linked to the N-terminus of IL-7. The A is characterized in that it contains 1 to 10 amino acids, and the amino acid may be selected from the group consisting of methionine, glycine, and combinations thereof.

Methionine and glycine do not induce an immune response in the body. Protein therapeutics produced from E. coli necessarily contain methionine at the N-terminus, but no immune side effects have been reported. In addition, glycine is widely used in GS linkers, and it does not induce an immune response even in commercially available products such as Dulaglutide ( Cell Biophys. 1993 Jan-Jun:22(1-3): 189- 224).

According to one embodiment, A may be an oligopeptide containing 1 to 10 amino acids selected from the group consisting of methionine (Met, M), glycine (Gly, G), and combinations thereof. Preferably, it may be an oligopeptide composed of 1 to 5 amino acids. For example, A is methionine, glycine, methionine-methionine, glycine-glycine, methionine-glycine, glycine-methionine, methionine-methionine-methionine, methionine-methionine-glycine, methionine-glycine-methionine, glycine-methionine-methionine , Methionine-glycine-glycine, glycine-methionine-glycine, glycine-glycine-methionine, and glycine-glycine-glycine may have one N-terminal sequence selected from the group consisting of. Specifically, A is methionine, glycine, methionine-methionine, glycine-glycine, methionine-glycine, glycine-methionine, methionine-methionine-methionine, methionine-methionine-glycine, methionine-glycine-methionine, glycine-methionine-methionine, It may be represented by an amino acid sequence selected from the group consisting of methionine-glycine-glycine, glycine-methionine-glycine, glycine-glycine-methionine, and glycine-glycine-glycine.

As used herein, the term "IL-2" or "Interleukin-2", unless otherwise stated, includes mammals such as primates (eg humans) and rodents (eg mice and rats). Refers to any wild-type IL-2 obtained from any vertebrate source. The term includes untreated IL-2, and any form of IL-2 obtained from treatment in cells. In addition, the term includes wild-type variants of IL-2, such as splice variants or allelic variants. Specifically, the amino acid sequence of human IL-2 may be SEQ ID NO: 7.

As used herein, the term "mutant IL-2" includes a truncated form of IL-2, and a form in which IL-2 is linked to another molecule by fusion or chemical conjugation, It can include any of the mutant forms in a variety of forms. Further, the mutant IL-2 may be a mutant obtained by substituting a portion of the amino acid of wild-type IL-2. At this time, the mutant IL-2 may be modified to have low toxicity in vivo. At this time, toxicity in vivo may mean a side effect caused by binding of IL-2 with the alpha chain of the IL-2 receptor. In order to attenuate the side effects of IL-2, various mutant IL-2s have been developed, and these mutant IL-2s are as disclosed in US Patent 5,229,109 and Korean Patent 1667096.

In the mutant IL-2, the 38th or 42nd amino acid of SEQ ID NO: 7 may be substituted. Specifically, one specific example of the mutant IL-2 may be a wild-type 38 th arginine substituted (R38A) with alanine. In addition, one specific example of the IL-2 variant may be a form in which the wild-type 42nd phenylalanine is substituted with another amino acid, and specifically, the 42nd amino acid of the sequence of SEQ ID NO: 7 is F42A, F42G, F42S, F42T, F42Q, F42E, It may be substituted with F42N, F42D, F42R and F42K.

In addition, the mutant IL-2 may be a form in which the 38th and 42nd amino acids are substituted in wild-type IL-2. For example, the mutant IL-2 may include any one amino acid substitution selected from R38A, F42A, and combinations thereof in the amino acid sequence represented by SEQ ID NO: 7. The mutant IL-2 may have the amino acid sequence of SEQ ID NO: 8.

The modified IL-7 and IL-2 can be bound by an immunoglobulin Fc domain.

The Fc domain may be wild type or variant. The Fc domain variant may be an Fc domain of a modified immunoglobulin. At this time, the Fc domain of the modified immunoglobulin is modified with the binding power to the Fc receptor and/or complement, so that antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) This could be weakened. The modified immunoglobulin may be selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and combinations thereof. In particular, the Fc domain of the modified immunoglobulin may include a hinge region, a CH2 domain, and a CH3 domain in the N-terminal to C-terminal direction. In this case, the hinge region includes a human IgD hinge region, and the CH2 domain includes a portion of an amino acid residue of a CH2 domain of human IgD and a portion of an amino acid residue of a CH2 domain of human IgG4, and the CH3 domain is of human IgG4. It may comprise a portion of the amino acid residues of the CH3 domain. The hinge region may be SEQ ID NO: 16.

As used herein, the term "Fc domain", "Fc fragment" or "Fc" includes heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3) of immunoglobulin, but variable regions of heavy and light chains thereof And light chain constant region 1 (CL1) refers to a protein that does not contain. It may further include a hinge region of the heavy chain constant region. Hybrid Fc or hybrid Fc fragment is also referred to herein as “hFc” or “hyFc”.

The term "Fc domain variant" used in the present invention means that some amino acids in the Fc domain are substituted or are prepared by combining different types of Fc domains. The Fc domain variant can prevent cleavage at the hinge region. Specifically, the 144th amino acid and/or the 145th amino acid of SEQ ID NO: 10 may be modified. Preferably, it may be a variant in which K, which is the 144th amino acid of SEQ ID NO: 10, is substituted with G or S, and E, which is the 145th amino acid, is substituted with G or S.

In addition, the Fc domain or Fc domain variant of the modified immunoglobulin can be represented by the following formula (I):

[Equation (I)]

N'-(Z1)p-Y-Z2-Z3-Z4-C'

In the above formula,

N'is the N-terminus of the polypeptide and C'is the C-terminus of the polypeptide;

p is an integer of 0 or 1; And

Z1 is an amino acid sequence having 5 to 9 consecutive amino acid residues in the N-terminal direction from the 98 position among the amino acid residues at positions 90 to 98 of SEQ ID NO: 11,

Y is an amino acid sequence having 5 to 64 consecutive amino acid residues in the N-terminal direction from position 162 among the amino acid residues at positions 99 to 162 of SEQ ID NO: 11,

Z2 is an amino acid sequence having 4 to 37 consecutive amino acid residues in the C-terminal direction from position 163 of the amino acid residues at positions 163 to 199 of SEQ ID NO: 11,

Z3 is an amino acid sequence having 71 to 106 contiguous amino acid residues in the N-terminal direction from the 220 position among the amino acid residues at positions 115 to 220 of SEQ ID NO: 12,

Z4 is an amino acid sequence having an amino acid sequence of 80 to 107 in the C-terminal direction from position 221 among the amino acid residues at positions 221 to 327 of SEQ ID NO: 12.

In addition, the Fc fragment of the present invention may be a natural type sugar chain, an increased sugar chain compared to the natural type, a reduced sugar chain compared to the natural type, or a form in which the sugar chain has been removed. The immunoglobulin Fc sugar chain can be modified by conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. Removal of the sugar chain from the Fc fragment sharply decreases the binding affinity of the primary complement component C1 to C1q, leads to a decrease or loss of ADCC or CDC, and thereby does not induce an unnecessary immune response in vivo. In this respect, the immunoglobulin Fc fragment in the form of deglycosylated or aglycosylated sugar chain may be more suitable for the purposes of the present invention as a drug carrier. The term "deglycosylation" as used herein means that sugars are enzymatically removed from an Fc fragment. In addition, the term "aglycosylation" means that the Fc fragment is produced in an unglycosylated form by a prokaryote, preferably E. coli.

In addition, the Fc domain of the modified immunoglobulin includes the amino acid sequence of SEQ ID NO: 10 (hyFc), SEQ ID NO: 5 (hyFcM1), SEQ ID NO: 13 (hyFcM2), SEQ ID NO: 14 (hyFcM3) or SEQ ID NO: 15 (hyFcM4) can do. In addition, the Fc domain of the modified immunoglobulin may include the amino acid sequence of SEQ ID NO: 5 (non-lytic mouse Fc).

According to the present invention, the Fc domain of the modified immunoglobulin may be described in U.S. Patent No. 7,867,491, and the production of the Fc domain of the modified immunoglobulin may be performed with reference to the bar described in U.S. Patent No. 7,867,491. .

In addition, the fusion protein may be a modified IL-7, Fc domain (hyFc), and IL-2 are sequentially bound from the N-terminus to the C-terminus. This fusion protein may be referred to as "modified IL-7-hyFc-IL-2v".

At this time, a first linker may be further included between the modified IL-7 and the Fc domain. The first linker may consist of 20 to 60 contiguous amino acids, or 25 to 50 contiguous amino acids, or 30 to 40 amino acids. In one embodiment, the first linker may consist of 15 amino acids. In addition, the first linker may include (G4S)n (here, n is an integer of 1 to 5). Preferably, the first linker may include an amino acid sequence represented by SEQ ID NO: 6.

In addition, a second linker may be further included between the Fc domain and IL-2.

The second linker may consist of 1 to 30 contiguous amino acids, or 3 to 20 contiguous amino acids, or 4 to 15 amino acids. In an embodiment, the second linker may be the amino acid sequence of SEQ ID NO: 17.

The fusion protein may have an amino acid sequence of SEQ ID NO: 9. In addition, the fusion protein is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 in the amino acid sequence of SEQ ID NO: 9 %, or 99% homology.

Another aspect of the present invention provides a pharmaceutical composition for the treatment or prevention of cancer or infectious diseases comprising a fusion protein containing modified IL-7 and IL-2 as an active ingredient.

At this time, the cancer is gastric cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myelogenous leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer and lymphoma. It may be any one selected from the group consisting of. In addition, the infectious disease may be any one selected from the group consisting of hepatitis B, hepatitis C, human papilloma virus (HPV) infection, cytomegalovirus infection, viral respiratory disease, and influenza. have.

In the composition for treating or preventing cancer or infectious diseases of the present invention, the active ingredient may be in an arbitrary amount (effective amount) according to the use, formulation, purpose of combination, etc., as long as it exhibits anti-cancer activity or can exhibit a therapeutic effect on infectious diseases. It may be included, and a typical effective amount will be determined within the range of 0.001% to 20.0% by weight based on the total weight of the composition. Here, "effective amount" refers to an amount of an active ingredient capable of inducing an anticancer effect or an infectious disease treatment effect. Such effective amounts can be determined empirically within the range of ordinary skill in the art.

In this case, the pharmaceutical composition may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be any carrier as long as it is a non-toxic material suitable for delivery to a patient. Distilled water, alcohols, fats, waxes and inert solids may be included as carriers. Pharmaceutically acceptable adjuvants (buffers, dispersants) may also be included in the pharmaceutical composition.

Specifically, the pharmaceutical composition may be prepared in a parenteral formulation according to an administration route by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. Here, "pharmaceutically acceptable" means that the application (prescription) does not have toxicity beyond adaptable without inhibiting the activity of the active ingredient.

When the pharmaceutical composition is prepared in a parenteral formulation, it may be formulated in the form of an injection, a transdermal administration, a nasal inhalation, and a suppository according to a method known in the art together with a suitable carrier. When formulated as an injection, sterile water, ethanol, polyols such as glycerol or propylene glycol, or a mixture thereof may be used as suitable carriers, preferably Ringer's solution, PBS (phosphate buffered saline) containing triethanol amine, or sterile water for injection. , An isotonic solution such as 5% dextrose may be used. Regarding the formulation of a pharmaceutical composition, it is known in the art, and specifically, reference may be made to Remington's Pharmaceutical Sciences (19th ed., 1995). This document is considered as part of this specification.

The preferred dosage of the pharmaceutical composition is in the range of 0.01 ug/kg to 10 g/kg per day, or 0.01 mg/kg to 1 g/kg per day depending on the patient's condition, weight, sex, age, patient severity, and route of administration. Can be Administration can be made once a day or divided into several times. Such dosage should not be construed as limiting the scope of the present invention in any aspect.

Subjects to which the pharmaceutical composition can be applied (prescribed) are mammals and humans, particularly preferably humans. In addition to the active ingredient, the pharmaceutical composition of the present application may further include any compound or natural extract, which has already been verified for safety and has a therapeutic effect on anticancer activity or infectious disease in order to increase and reinforce anticancer activity.

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited thereto.

Example 1. Modified IL7-hyFc-IL2v Gene Construct Preparation

In order to prepare a fusion protein in which human-derived modified IL-7 and IL-2 mutant (IL-2v) are bound, as shown in FIG. 1, IL-7 from the N-terminus to the C-terminus, the first linker , An Fc domain, a second linker, and a gene construct in the form of binding in the order of IL-2v was prepared.

Specifically, in order to prepare an expression vector containing a modified human-derived IL7-hyFc-IL2v structural gene, the modified IL-7 is a modified IL-7 in which an oligopeptide is bound to the N-terminus of human IL-7. Was used. The human IL-7 used the amino acid sequence represented by SEQ ID NO: 1, and the oligopeptide used the amino acid sequence represented by SEQ ID NO: 2. Specifically, the modified IL-7 has an amino acid sequence represented by SEQ ID NO: 3. In addition, it has an Fc domain comprising hyFc consisting of a hinge region of SEQ ID NO: 16 and an amino acid sequence represented by SEQ ID NO: 5. The C-terminus of the modified IL-7 and the N-terminus of the Fc domain were bound through a first linker consisting of an amino acid sequence represented by SEQ ID NO: 6. In addition, the C-terminus of the Fc domain and the N-terminus of IL-2v were bound through a second linker represented by SEQ ID NO: 17. In the case of IL-2v, a sequence containing two mutant regions that is expected to decrease the binding ability to IL-2 receptor α and increase therapeutic efficacy through selective binding to NK cells was used. Specifically, the IL-2v was used as having amino acid substitutions of R38A and F42A in human IL-2 (SEQ ID NO: 7), and consisting of the amino acid sequence represented by SEQ ID NO: 8.

A final gene construct was prepared through the above sequences to obtain a recombinant protein expression vector. The modified IL7-hyFc-IL2v gene synthesized each gene and prepared it as a sub-vector, and then proceeded to Golden Gateway assembly to prepare the synthesized three gene segments into one gene segment, and the expression vector was converted into a pBispec vector. Secured.

Expression vector was transfected ^{into suspension-adapted Chinese Hamster Ovary (CHO) cells using ExpiCHO TM} Expression System (Thermo Fisher, A29133), cultured for 7 days, harvested, and purified to obtain proteins.

Example 2. Production of modified IL7-hyFc-IL2v fusion protein

In order to produce the modified IL7-hyFc-IL2v fusion protein of Example 1, suspension-adapted CHO cells ^{were inoculated at 6×10 6} cells/ml, and the expression vector prepared in Example 1 was 600 ml of ExpiFectamine ^TM Transformation was carried out using CHO reagent. To secure a large amount of protein, ExpiFectamine ^TM CHO Enhancer and ExpiCHO ^TM Feed were added the day after transformation, and a shift was performed at a temperature of 32°C. After 7 days of culture, the culture medium was collected, centrifuged at 3,600 rpm for 15 minutes to separate the supernatant, and then only the supernatant was obtained using a 0.22 um bottle top filter.

To purify the protein, impurities other than the modified IL7-hyFc-IL2v fusion protein were removed through Protein A resin using AKTA Prime (GE Healthcare Life sciences). After that, the target protein was eluted with an elution buffer (Elution buffer: 50mM Glycine-HCl, 500mM L-Arginine, 300mM NaCl, pH 4.1) through Protein A resin, and then confirmed through SDS-PAGE (Fig. 2).

As a result, a protein having a purity of 90% or more could be secured, and only a fraction having a high purity was separated, and formulation was performed with PBS (pH 7.4).

After formulation, the modified IL7-hyFc-IL2v fusion protein was confirmed by SDS-PAGE analysis, and as a result, it was confirmed at about 122 kDa in a non-reducing condition, and about 50 to about 50 in a reducing condition. It was identified at 75 kDa. In addition, as a result of SE-HPLC analysis, a purity of 91% was confirmed (FIG. 3).

Example 3. Comparison of the activity of the modified IL7-hyFc-IL2v fusion protein: in vitro

The activity analysis of the modified IL7-hyFc-IL2v fusion protein was performed using immune cells extracted from the lymph nodes of C57BL/6 mice. Before inoculating the immune cells, in order to check the proliferation rate, CellTrace?? at a concentration of 5 μM It was stained using Violet (Invitrogen??). After inoculating a 96-well plate so that the stained immune cells were 1×10 ⁵ cells/50 μl per well, each well was treated with an anti-CD3ε antibody at a concentration of 8 ng/ml, and the modified IL7-hyFc and The modified IL7-hyFc-IL2v fusion protein was treated at a concentration of 10 ng/ml and incubated at 37°C and 5% CO ₂ for 72 hours. Since then, the BD LSRFortessa?? The proliferation rate of CD8+ T cells was measured using a flow cytometer.

As a result, in the case of CD8+ T cells treated with the modified IL7-hyFc-IL2v fusion protein, it was confirmed that the proliferation rate was improved compared to the negative control (Anti-CD3ε alone) and the positive control (IL7-hyFc) (Fig. 4). .

In addition, the expression levels of intracellular anticancer-related cytokines and cytolytic enzymes Granzyme B (GzmB), IFN-gamma (IFNγ), and TNF-alpha (TNFα) in the CD8+ T cells were also measured using intracellular staining (ICS). It was measured.

As a result, in the case of CD8+ T cells treated with the modified IL7-hyFc-IL2v fusion protein, Granzyme B, IFN-gamma, and TNF-alpha were compared to the negative control (Anti-CD3ε alone) and the positive control (IL7-hyFc). It was confirmed that the expression level was increased (FIG. 5).

Example 4. Comparison of the activity of the modified IL7-hyFc-IL2v fusion protein: in vivo

In vivo activity analysis was performed using immune cells extracted from the spleen of a mouse with a CD45.1-like genotype. CellTrace at a concentration of 5 μM in CD45.2 C57BL/6 mice?? Violet-stained 1×10 ⁷ cells/mouse of immune cells were administered intravenously. One day later, PBS, modified IL7-hyFc, and modified IL7-hyFc-IL2v fusion protein (10 mg/kg) were administered by subcutaneous injection, and 5 days after administration, the spleen of the mouse was excised and the degree of differentiation of CD8+ T cells. BD LSRFortessa?? It was confirmed using a flow cytometer.

As a result, it was confirmed that when the modified IL7-hyFc-IL2v fusion protein was administered, the differentiation of CD8+ T cells was promoted compared to the negative control (PBS) (FIG. 6).

Example 5. Comparison of anticancer efficacy of modified IL7-hyFc-IL2v in colon cancer cell line transplanted mice

Colon cancer cell line transplantation mice were used to confirm the anticancer efficacy and changes in immune cells of the modified IL7-hyFc-IL2v fusion protein.

After transplanting the MC38 cancer cell line, a colon cancer cell line derived from a mouse of 1×10 ⁵ cells, into the right flank of the mouse, PBS, modified IL7-hyFc, and modified IL7-hyFc-IL2v fusion protein (10 mg/kg) were added. It was administered subcutaneously once, and the size of the cancer was measured over time. In addition, changes in blood immune cells obtained by cross-bleeding between individuals in the group were analyzed through flow cytometry.

As a result, in the case of mice administered with the modified IL7-hyFc-IL2v fusion protein, the size of the cancer was significantly suppressed compared to the negative control (PBS), and showed a similar cancer inhibitory effect to the positive control (modified IL7-hyFc) Confirmed. In addition, it was confirmed that differentiation from immune cells in blood of mice administered with the modified IL7-hyFc-IL2v fusion protein to NK cells was promoted (FIG. 7).

Through this, it is determined that the modified IL7-hyFc exhibits anticancer ability based on the specific differentiation ability of CD8+ T cells, but the modified IL7-hyFc-IL2v fusion protein shows excellent differentiation ability into NK cells, while also modifying the anticancer effect. It can be seen that the IL7-hyFc exhibited similar levels.

<110> Genexine, Inc. <120> FUSION PROTEIN COMPRISING MODIFIED INTERLEUKIN-7 AND INTERLEUKIN-2 AND USE THEREOF <130> FPD/201911-0006 <160> 17 <170> KoPatentIn 3.0 <210> 1 <211> 177 <212> PRT <213> Homo sapiens <400> 1 Met Phe His Val Ser Phe Arg Tyr Ile Phe Gly Leu Pro Pro Leu Ile 1 5 10 15 Leu Val Leu Leu Pro Val Ala Ser Ser Asp Cys Asp Ile Glu Gly Lys 20 25 30 Asp Gly Lys Gln Tyr Glu Ser Val Leu Met Val Ser Ile Asp Gln Leu 35 40 45 Leu Asp Ser Met Lys Glu Ile Gly Ser Asn Cys Leu Asn Asn Glu Phe 50 55 60 Asn Phe Phe Lys Arg His Ile Cys Asp Ala Asn Lys Glu Gly Met Phe 65 70 75 80 Leu Phe Arg Ala Ala Arg Lys Leu Arg Gln Phe Leu Lys Met Asn Ser 85 90 95 Thr Gly Asp Phe Asp Leu His Leu Leu Lys Val Ser Glu Gly Thr Thr 100 105 110 Ile Leu Leu Asn Cys Thr Gly Gln Val Lys Gly Arg Lys Pro Ala Ala 115 120 125 Leu Gly Glu Ala Gln Pro Thr Lys Ser Leu Glu Glu Asn Lys Ser Leu 130 135 140 Lys Glu Gln Lys Lys Leu Asn Asp Leu Cys Phe Leu Lys Arg Leu Leu 145 150 155 160 Gln Glu Ile Lys Thr Cys Trp Asn Lys Ile Leu Met Gly Thr Lys Glu 165 170 175 His <210> 2 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for A (MGM) <400> 2 Met Gly Met One <210> 3 <211> 180 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for modified IL-7 (signal peptide-MGM-IL-7) <400> 3 Met Phe His Val Ser Phe Arg Tyr Ile Phe Gly Leu Pro Pro Leu Ile 1 5 10 15 Leu Val Leu Leu Pro Val Ala Ser Ser Met Gly Met Asp Cys Asp Ile 20 25 30 Glu Gly Lys Asp Gly Lys Gln Tyr Glu Ser Val Leu Met Val Ser Ile 35 40 45 Asp Gln Leu Leu Asp Ser Met Lys Glu Ile Gly Ser Asn Cys Leu Asn 50 55 60 Asn Glu Phe Asn Phe Phe Lys Arg His Ile Cys Asp Ala Asn Lys Glu 65 70 75 80 Gly Met Phe Leu Phe Arg Ala Ala Arg Lys Leu Arg Gln Phe Leu Lys 85 90 95 Met Asn Ser Thr Gly Asp Phe Asp Leu His Leu Leu Lys Val Ser Glu 100 105 110 Gly Thr Thr Ile Leu Leu Asn Cys Thr Gly Gln Val Lys Gly Arg Lys 115 120 125 Pro Ala Ala Leu Gly Glu Ala Gln Pro Thr Lys Ser Leu Glu Glu Asn 130 135 140 Lys Ser Leu Lys Glu Gln Lys Lys Leu Asn Asp Leu Cys Phe Leu Lys 145 150 155 160 Arg Leu Leu Gln Glu Ile Lys Thr Cys Trp Asn Lys Ile Leu Met Gly 165 170 175 Thr Lys Glu His 180 <210> 4 <211> 155 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for modified IL-7(MGM-IL-7) <400> 4 Met Gly Met Asp Cys Asp Ile Glu Gly Lys Asp Gly Lys Gln Tyr Glu 1 5 10 15 Ser Val Leu Met Val Ser Ile Asp Gln Leu Leu Asp Ser Met Lys Glu 20 25 30 Ile Gly Ser Asn Cys Leu Asn Asn Glu Phe Asn Phe Phe Lys Arg His 35 40 45 Ile Cys Asp Ala Asn Lys Glu Gly Met Phe Leu Phe Arg Ala Ala Arg 50 55 60 Lys Leu Arg Gln Phe Leu Lys Met Asn Ser Thr Gly Asp Phe Asp Leu 65 70 75 80 His Leu Leu Lys Val Ser Glu Gly Thr Thr Ile Leu Leu Asn Cys Thr 85 90 95 Gly Gln Val Lys Gly Arg Lys Pro Ala Ala Leu Gly Glu Ala Gln Pro 100 105 110 Thr Lys Ser Leu Glu Glu Asn Lys Ser Leu Lys Glu Gln Lys Lys Leu 115 120 125 Asn Asp Leu Cys Phe Leu Lys Arg Leu Leu Gln Glu Ile Lys Thr Cys 130 135 140 Trp Asn Lys Ile Leu Met Gly Thr Lys Glu His 145 150 155 <210> 5 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for hyFcM1 <400> 5 Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys 1 5 10 15 Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 20 25 30 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 35 40 45 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 50 55 60 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 65 70 75 80 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 85 90 95 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 100 105 110 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 115 120 125 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 130 135 140 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 145 150 155 160 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 165 170 175 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 180 185 190 Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 195 200 205 Leu Ser Leu Ser Leu Gly Lys 210 215 <210> 6 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for linker 1 <400> 6 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 7 <211> 133 <212> PRT <213> Homo sapiens <400> 7 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 8 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for IL-2v <400> 8 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Ala Met Leu Thr Ala Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 9 <211> 562 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for IL7-hyFc-IL2v <400> 9 Met Phe His Val Ser Phe Arg Tyr Ile Phe Gly Leu Pro Pro Leu Ile 1 5 10 15 Leu Val Leu Leu Pro Val Ala Ser Ser Met Gly Met Asp Cys Asp Ile 20 25 30 Glu Gly Lys Asp Gly Lys Gln Tyr Glu Ser Val Leu Met Val Ser Ile 35 40 45 Asp Gln Leu Leu Asp Ser Met Lys Glu Ile Gly Ser Asn Cys Leu Asn 50 55 60 Asn Glu Phe Asn Phe Phe Lys Arg His Ile Cys Asp Ala Asn Lys Glu 65 70 75 80 Gly Met Phe Leu Phe Arg Ala Ala Arg Lys Leu Arg Gln Phe Leu Lys 85 90 95 Met Asn Ser Thr Gly Asp Phe Asp Leu His Leu Leu Lys Val Ser Glu 100 105 110 Gly Thr Thr Ile Leu Leu Asn Cys Thr Gly Gln Val Lys Gly Arg Lys 115 120 125 Pro Ala Ala Leu Gly Glu Ala Gln Pro Thr Lys Ser Leu Glu Glu Asn 130 135 140 Lys Ser Leu Lys Glu Gln Lys Lys Leu Asn Asp Leu Cys Phe Leu Lys 145 150 155 160 Arg Leu Leu Gln Glu Ile Lys Thr Cys Trp Asn Lys Ile Leu Met Gly 165 170 175 Thr Lys Glu His Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 180 185 190 Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 195 200 205 Cys Pro Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys 210 215 220 Pro Lys Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 225 230 235 240 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 245 250 255 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 260 265 270 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 275 280 285 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 290 295 300 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 305 310 315 320 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 325 330 335 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 340 345 350 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 355 360 365 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 370 375 380 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 385 390 395 400 Phe Ser Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln 405 410 415 Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Gly Gly Ser Ala Pro Thr 420 425 430 Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu 435 440 445 Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys 450 455 460 Leu Thr Ala Met Leu Thr Ala Lys Phe Tyr Met Pro Lys Lys Ala Thr 465 470 475 480 Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu 485 490 495 Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg 500 505 510 Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser 515 520 525 Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val 530 535 540 Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr 545 550 555 560 Leu Thr <210> 10 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for hyFc <400> 10 Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys 1 5 10 15 Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His 20 25 30 Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 35 40 45 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 50 55 60 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 65 70 75 80 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 85 90 95 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 100 105 110 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 115 120 125 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 130 135 140 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 145 150 155 160 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 165 170 175 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 180 185 190 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 195 200 205 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 210 215 220 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 225 230 235 240 Leu Ser Leu Gly Lys 245 <210> 11 <211> 384 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for human IgD constant region (Genbank accession No. P01880) <400> 11 Ala Pro Thr Lys Ala Pro Asp Val Phe Pro Ile Ile Ser Gly Cys Arg 1 5 10 15 His Pro Lys Asp Asn Ser Pro Val Val Leu Ala Cys Leu Ile Thr Gly 20 25 30 Tyr His Pro Thr Ser Val Thr Val Thr Trp Tyr Met Gly Thr Gln Ser 35 40 45 Gln Pro Gln Arg Thr Phe Pro Glu Ile Gln Arg Arg Asp Ser Tyr Tyr 50 55 60 Met Thr Ser Ser Gln Leu Ser Thr Pro Leu Gln Gln Trp Arg Gln Gly 65 70 75 80 Glu Tyr Lys Cys Val Val Gln His Thr Ala Ser Lys Ser Lys Lys Glu 85 90 95 Ile Phe Arg Trp Pro Glu Ser Pro Lys Ala Gln Ala Ser Ser Val Pro 100 105 110 Thr Ala Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala 115 120 125 Pro Ala Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys 130 135 140 Glu Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu 145 150 155 160 Cys Pro Ser His Thr Gln Pro Leu Gly Val Tyr Leu Leu Thr Pro Ala 165 170 175 Val Gln Asp Leu Trp Leu Arg Asp Lys Ala Thr Phe Thr Cys Phe Val 180 185 190 Val Gly Ser Asp Leu Lys Asp Ala His Leu Thr Trp Glu Val Ala Gly 195 200 205 Lys Val Pro Thr Gly Gly Val Glu Glu Gly Leu Leu Glu Arg His Ser 210 215 220 Asn Gly Ser Gln Ser Gln His Ser Arg Leu Thr Leu Pro Arg Ser Leu 225 230 235 240 Trp Asn Ala Gly Thr Ser Val Thr Cys Thr Leu Asn His Pro Ser Leu 245 250 255 Pro Pro Gln Arg Leu Met Ala Leu Arg Glu Pro Ala Ala Gln Ala Pro 260 265 270 Val Lys Leu Ser Leu Asn Leu Leu Ala Ser Ser Asp Pro Pro Glu Ala 275 280 285 Ala Ser Trp Leu Leu Cys Glu Val Ser Gly Phe Ser Pro Pro Asn Ile 290 295 300 Leu Leu Met Trp Leu Glu Asp Gln Arg Glu Val Asn Thr Ser Gly Phe 305 310 315 320 Ala Pro Ala Arg Pro Pro Pro Gln Pro Gly Ser Thr Thr Phe Trp Ala 325 330 335 Trp Ser Val Leu Arg Val Pro Ala Pro Pro Ser Pro Gln Pro Ala Thr 340 345 350 Tyr Thr Cys Val Val Ser His Glu Asp Ser Arg Thr Leu Leu Asn Ala 355 360 365 Ser Arg Ser Leu Glu Val Ser Tyr Val Thr Asp His Gly Pro Met Lys 370 375 380 <210> 12 <211> 327 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for Partial human IgG4 constant region (Genbank accession No. AAH25985) <400> 12 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 13 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for hyFcM2 <400> 13 Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Gly Ser Lys Glu Lys 1 5 10 15 Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His 20 25 30 Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 35 40 45 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 50 55 60 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 65 70 75 80 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 85 90 95 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 100 105 110 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 115 120 125 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 130 135 140 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 145 150 155 160 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 165 170 175 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 180 185 190 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 195 200 205 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 210 215 220 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 225 230 235 240 Leu Ser Leu Gly Lys 245 <210> 14 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for hyFcM3 <400> 14 Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Ser Gly Lys Glu Lys 1 5 10 15 Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His 20 25 30 Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 35 40 45 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 50 55 60 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 65 70 75 80 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 85 90 95 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 100 105 110 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 115 120 125 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 130 135 140 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 145 150 155 160 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 165 170 175 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 180 185 190 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 195 200 205 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 210 215 220 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 225 230 235 240 Leu Ser Leu Gly Lys 245 <210> 15 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for hyFcM4 <400> 15 Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Ser Ser Lys Glu Lys 1 5 10 15 Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His 20 25 30 Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 35 40 45 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 50 55 60 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 65 70 75 80 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 85 90 95 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 100 105 110 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 115 120 125 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 130 135 140 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 145 150 155 160 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 165 170 175 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 180 185 190 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 195 200 205 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 210 215 220 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 225 230 235 240 Leu Ser Leu Gly Lys 245 <210> 16 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for IgG1 hinge <400> 16 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 <210> 17 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for linker 2 <400> 17 Gly Gly Gly Ser One

Claims (19)

Fusion protein comprising modified interleukin-7 (IL-7) and interleukin-2 (IL-2). The method of claim 1,
The modified IL-7 will have the following structure, a fusion protein:
A-IL-7
Wherein A is an oligopeptide consisting of 1 to 10 amino acid residues (provided that A is not methionine),
The modified IL-7 is IL-7 or a polypeptide having similar activity. The method of claim 2,
The IL-7 is a fusion protein consisting of the amino acid sequence represented by SEQ ID NO: 1. The method of claim 2,
A fusion protein that is linked to the N-terminus of IL-7. The method of claim 2,
A is glycine, methionine-methionine, glycine-glycine, methionine-glycine, glycine-methionine, methionine-methionine-methionine, methionine-methionine-glycine, methionine-glycine-methionine, glycine-methionine-methionine, methionine-glycine-glycine , Glycine-methionine-glycine, glycine-glycine-methionine, and any one selected from the group consisting of glycine-glycine-glycine, a fusion protein. The method of claim 1,
The IL-2 is a fusion protein consisting of the amino acid sequence represented by SEQ ID NO: 7. The method of claim 1,
The IL-2 is a mutant IL-2, a fusion protein. The method of claim 7,
The mutant IL-2 is a fusion protein comprising any one amino acid substitution selected from the group consisting of R38A, F42A, and combinations thereof in the amino acid sequence represented by SEQ ID NO: 7. The method of claim 8,
The mutant IL-2 is a fusion protein consisting of the amino acid sequence represented by SEQ ID NO: 8. The method of claim 1,
The modified IL-7 and IL-2 are bound by an immunoglobulin Fc domain, a fusion protein. The method of claim 10,
The Fc domain is a wild type or a mutant, a fusion protein. The method of claim 10,
The modified IL-7, Fc domain and IL-2 are bound in order from the N-terminus to the C-terminus, a fusion protein. The method of claim 12,
A fusion protein that further comprises a first linker between the modified IL-7 and the Fc domain. The method of claim 13,
The first linker is consisting of the amino acid sequence represented by SEQ ID NO: 6, fusion protein. The method of claim 12,
A fusion protein that further comprises a second linker between the Fc domain and IL-2. The method of claim 15,
The second linker is a fusion protein consisting of the amino acid sequence represented by SEQ ID NO: 17. A pharmaceutical composition for preventing or treating cancer or infectious diseases comprising the fusion protein according to any one of claims 1 to 16 as an active ingredient. The method of claim 17,
The cancer is gastric cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myelogenous leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer and lymphoma. Any one selected from the group, a pharmaceutical composition. The method of claim 17,
The infectious disease is any one selected from the group consisting of hepatitis B, hepatitis C, human papilloma virus infection, cytomegalovirus infection, viral respiratory disease and influenza, a pharmaceutical composition.

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